The use of vaccines likely represents the most important, practical contribution that immunologists have made to human health. Effective vaccines establish a state of resistance to microbial diseases in the host prior to infection. Thus, vaccinations function to prevent disease, and such prophylaxis has undoubtedly saved countless lives. In fact, vaccinations have been so successful, that widespread immunizations directed against a variety of bacterial, viral, and parasitic pathogens have become the rule, once an appropriate vaccine has been identified. The most notable example of global immunization was the use of a vaccine against smallpox which eliminated this viral disease.
Ultimately, the success and practicality of widespread immunizations against a particular pathogen depends upon the characteristics of an individual vaccine. These characteristics include: 1) efficacy, i.e. the ability to induce a response that imparts some level of protection in the vast majority of individuals; 2) safety, i.e. limited side effects in immunocompetent children and/or adults; 3) method of delivery, e.g. injectable, oral, intranasal, or transdermal administration; 4) immunization regimen, i.e. single or multiple exposures; 5) stability, i.e., the shelf life and conditions needed for shipping, such as refrigeration; and 6) cost, i.e., the total expense for an immunization regimen, including vaccine production, shipping, and administration. Using these considerations, the most successful vaccines would likely be cheap to produce, highly stable for shipping, and administered in a desirable form that does not require specialized personnel for delivery.
The enormous potential for the use of plant-derived vaccines has been discussed since the first demonstration of the feasibility of such technology in the early 1990s (Mason et al. 1992. Expression of hepatitis B surface antigen in transgenic plants. Proc Natl Acad Sci U S A 89:11745, which is herein incorporated by reference in its entirety). An edible vaccine expressed in transgenic plants would represent a cost-effective method for production, as well as the promise of safe administration of an antigen in a highly stable form that could be shipped throughout the world. While the concept of eating a vaccine is easy to visualize, this technology is relatively new. Although some progress has been made toward demonstrating the feasibility of expressing subunit protein antigens in plants, there are drawbacks to the systems methods and use as well as some practical questions concerning the effectiveness of edible vaccines that prior to the instant invention had yet to be addressed.
Despite the promise of plant based vaccines as a low cost method for stimulating mucosal immunity, significant questions still remain about the feasibility of developing such methods for immunizing humans and animals.
Moreover, while widespread vaccination to prevent microbial diseases occurs daily throughout the world, a similar, prophylactic approach has not been used for the widespread prevention of allergic reactions and/or autoimmune antigens. Presently, immunotherapy may be used once an individual has already demonstrated a significant hypersensitivity against a particular allergen or has developed a specific autoimmune disease. However, it has seemed impractical to suggest that it might be feasible to induce tolerance toward specific allergens in individuals even before they demonstrate hypersensitivity.
A criticism for all investigators who have attempted to express vaccines in transgenic plants has been the efficacy and practicality of such immunization strategies. Often the immunogen expressed in plants has had to be purified or significantly concentrated prior to its use as a vaccine. Moreover, often the concentrated or purified plant-derived immunogen must be given parenterally or intranasally to demonstrate its ability to stimulate an immune response.
Soybeans first emerged as a domestic crop in the eastern half of China around the 11th century. Soybeans were later introduced from the Orient to Europe in the early 1700s, and then to North America in the early 1800s. Large scale introduction in the US was in the early 1900s. (from “Soybeans Chemistry, Technology, and Utilization, by KeShun Liu, Aspen Publishers, Inc. Gaithersburg, Maryland, 1999, ISBN: 0-8342-1299-4). The incorporation of recombinant nucleotide sequences into soybeans first made an appearance in 1988 see Hinchee, et al. 1988. Bio/Technology 6, 915-922. However, to the inventors knowledge, to date, no transgenic soybeans have been used or exploited for antigen production, for soy milk formulations, for use to make adjuvants, for edible vaccines in general, or for any other immunogenic purpose.
1. Field of the Invention
The field of the present invention relates to vaccines that are made in transgenic soybeans for use in humans, animals of agricultural importance, pets, and wildlife. These vaccines can be used for a plurality of purposes, such as vaccines against viral, bacterial, fungal, parasitic or prion related diseases, cancer antigens, toxins, and autologous or self proteins. Moreover, the transgenic soybeans of the instant invention can be used for inducing tolerance to allergens or tolerance to autoimmune antigens, wherein an individual shows hypersensitivity to said allergen or has developed autoimmunity to autologous or self proteins, respectively. The present invention also relates to prophylatically treating individuals and/or populations prior to showing hypersensitivity to allergens. Other aspects of the invention include using the transgenic soybeans as an oral contraceptive, and the expression of protein adjuvants in transgenic soybeans.
2. Description of Related Art
U.S. Pat. Nos. 6,034,298 and 6,136,320, both of which are incorporated herein in their entirety, disclose using a hepatitis B surface antigen, which is incorporated into potato tubers to generate an immune response. Potato tubers are known to have protein content, which is usually on the order of 2% (See http://beef.osu.edu/library/potato.html visited on Oct. 12, 2005 for a report on the protein content in potatoes). Soybeans, in contrast, have protein amounts that are on the order of 38% protein. The present invention generally has protein amounts that are equal to or above 15%, more preferably 20% and even more preferably 30%. Thus, it is noted that soybeans have more than an order of magnitude higher concentration of protein than potatoes. Because of this relatively low protein content in potatoes, one may or may not generate an immune response since it may or may not be possible to provide an effective dose without consumption of an unrealistic amount of raw potato. Generally, the immune response may be attributable to the concentration of the antigen protein, some unusual property of the antigen protein being expressed or the combinatorial use of an adjuvant to increase immune response. In U.S. Pat. Nos. 6,034,298 and 6,136,320, the immune response was triggered due to unusual properties of hepatitis B surface antigen. There are two properties that likely made the hepatic B subunit antigen immunogenic at the relatively low protein concentrations seen in potato tubers.
First, the hepatitis B subunit antigen self-associates into large polymers. This is an unusual property that is not possessed by most subunit protein antigens. In fact, the vast majority of subunit antigens (i.e. >95%) will not self-associate into such large conglomerates. It is known that large polymers are much more immunogenic than single proteins. Thus, for the hepatitis B subunit antigen, the immunogenic response was likely attained in part due to this self-association. In the absence of self association (or high concentrations), the probability of it being immunogenic when given orally would be negligible without the incorporation of an adjuvant used in conjunction with the protein antigen.
Second, the hepatitis B subunit antigen protein happens to bind to epithelial cells on mucosal surfaces. The vast majority of subunit antigens (i.e. >95%) will not have such a property. Therefore, most subunit antigens will pass right through the gut without ever interacting to any large extent with epithelial cells (unless the concentration is sufficiently high or unless the vaccine is combined with an adjuvant). Such antigens that don't bind to mucosal surfaces have a very low probability of being immunogenic without the incorporation of an adjuvant (or without having the concentration sufficiently high to overcome the poor immunogenic response). Thus, using potato or some other transgenic plant other than soybean is disadvantageous in that it generally requires the a subunit protein antigen to have some unusual property in order to get an immune response.
U.S. Pat. Nos. 6,034,298 and 6,136,320 also focus on “antigens located on the surface of a pathogenic organism”, which may be a limitation of the system (i.e., potatoes) that is used. These antigens only represent a small number of viral or bacterial antigens and they are not likely to include some of the more important antigens that can be used in vaccine development. A protein does not have to be on the surface of a pathogen to be immunogenic or to provide protective immunity.
Responses of T helper lymphocytes are triggered by antigens that do not have to appear on the surface of a pathogenic organism. Memory T helper cells must be formed to have optimal antibody responses and for optimal cytotoxic T cell responses. In fact, helper T lymphocytes cannot recognize antigens on the surface of pathogen. They can recognize peptide fragments from any protein antigen from the pathogen (i.e. external, cytoplasmic, nuclear, etc.). The reason for this is that T cells don't recognize antigens directly (like antibodies do), but rather T cells must recognize proteins that have been processed into peptides and expressed on an antigen presenting cells (i.e. dendritic cells or macrophages). T helper cells recognize peptide antigens presented to them by antigen presenting cells. Thus, one good measure of a vaccine is its “processcivity”, i.e., how well it can be degraded by antigen presenting cells so that it can be presented to T helper cells so they can help B lymphocytes and T cytotoxic cells perform their function. Thus, the consideration of antigens appearing (or not appearing) on the surface of a pathogenic organism is a relevant consideration to keep in mind when making a vaccine.
Similarly, T cytotoxic lymphocytes target and kill virally infected cells (without having to recognize antigens on the surface of a pathogenic organism). A primary goal of many anti-viral vaccines is to stimulate a cytotoxic T lymphocyte response to combat viral infections. T cytotoxic lymphocytes can only recognize viral peptides presented to them by the infected cell (i.e. epithelial cell) that the cytotoxic cells are trying to kill. Thus, any subunit antigen (i.e. external, internal, etc.) should be considered an appropriate vaccine candidate. The ability of a vaccine to stimulate T cytotoxic cell activity using subunit vaccines that are not expressed on the surface of the pathogen is an important consideration in designing a vaccine.
Moreover, the goal of oral vaccines is generally to induce the production of long lived T helper and B lymphocyte memory cells. Only if such memory cells are induced can a vaccine be efficacious. If a vaccine does not induce long lived memory cells, then it is likely to not be effective. Different vaccines induce different longevities of memory cells. For example, it is known that a tetanus immunization should be updated every decade or so, but the attenuate polio vaccine likely induces inmmunity for life. This is due to the nature of this particular vaccine's ability to induce very long-lived B and T memory cells. If one does not recognize these goals and drawbacks, one cannot effectively design a vaccine or propose assays to determine vaccine efficacy.
Another consideration that should be kept in mind when designing a vaccine is overcoming oral tolerance. Subunit antigens given orally which do not have special properties (i.e. high affinity for gut epithelium or endogenous adjuvant activity) will pass through the gastrointestinal tract without stimulating any detectable memory B or T lymphocyte formation. Thus, vaccine formulations which include most (>95%) of oral subunit protein vaccines must have a strategy to overcome oral tolerance. In the absence of such a strategy, it is highly unlikely that most subunit protein will function as a useful vaccine when given orally. One means of having a subunit vaccine given orally inducing long term immunity is by the concurrent administration of an adjuvant.
The ability of oral vaccine formulations to induce mucosal and systemic memory responses depends upon the expansion of memory T and B lymphocytes at mucosal and at systemic sites. Therefore, the goal of an effective mucosal immune response is not solely the formation of mucosal IgA, but includes the formation of memory T helper lymphocytes and the formation of memory T cytotoxic lymphocytes at mucosal and systemic sites. Failure to recognize this fact limits the scope of vaccine development when designing oral vaccine formulations.